This invention relates to nucleic acid and amino acid sequences of a human short-chain dehydrogenase and to the use of these sequences in the diagnosis, treatment, and prevention of immune disorders and cancer.
Acetyl CoA is a key intermediate in the mitochondrial metabolism of pyruvate and fatty acids. Pyruvate which is generated in the cytosol during glycolysis, is transported across the mitochondrial membranes to the interior mitochondrial matrix. The complete oxidation of pyruvate to form CO2 and H2O occurs in the mitochondrion and utilizes O2 as the final electron acceptor (oxidizer). Immediately on entering the matrix, pyruvate reacts with coenzyme A to form CO2 and the intermediate acetyl CoA, a reaction catalyzed by the enzyme pyruvate dehydrogenase. This reaction is highly exergonic (xcex94Gxc2x0=xe2x88x928.0 kcal/mol) and is essentially irreversible. Pyruvate dehydrogenase is one of the most complex enzymes known. The molecule is 30 nm in diameter and contains 60 subunits composed of three different enzymes, several regulatory polypeptides, and five different coenzymes. Fatty acids are also oxidized in the mitochondrion to produce acetyl CoA; the energy released is used to synthesize ATP form ADP and phosphate ion. In eucaryotic cells, fatty acids containing approximately 20 CH2 groups are degraded chiefly in peroxisomes and converted to acetyl CoA.
Fatty acids are stored as triglycerols, primarily as droplets in adipose cells. In response to hormones such as adrenaline, triglycerols are hydrolyzed in the cytosol to free fatty acids and glycerol. Fatty acids are released into the blood, where they are taken up and used by most cells. They are the major energy source for many tissues, in particular, heart muscle. In humans, the oxidation of fats is quantitatively more important than the oxidation of glucose as a source of ATP, due to the fact that oxidation of 1 gram of triacylglycerol to CO2 generates about six times as much ATP as does the oxidation of 1 gram of hydrated glycogen.
Nicotinamide adenine dinucleotides are involved in a very large number of oxidoreduction reactions both in the cytosol and in mitochondria, including the oxidation of the acetyl group of acetyl CoA to CO2. In general, nicotinamide adenine dinucleotides are not tightly bound to enzymes and may function as substrates, although they are often referred to as coenzymes. Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) undergo reversible reduction to NADH and NADPH, respectively, but have different activities in the cell. The major role of NADH is to transfer electrons from metabolic intermediates in a large number of biosynthetic processes into the electron transfer chain. NADPH acts as a reducing agent in a large number of biosynthetic processes.
In the cytosol, free fatty acids are linked to coenzyme A to form an acyl CoA in an energetic reaction coupled to the hydrolysis of ATP to AMP and inorganic pyrophosphate. The fatty acyl group is then transported across the inner mitochondrial membrane by a transporter protein and is reattached to another CoA molecule on the matrix side. Each molecule of acyl CoA in the mitochondrion is oxidized to form one molecule of acetyl CoA and an acyl CoA shortened by two carbon atoms. Concomitantly, NAD+ and FAD are reduced to NADH and FADH2, respectively. This set of reactions is repeated on the shortened acyl CoA until all carbon atoms are converted to acetyl CoA. Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid beta-oxidation. Defects in the SCAD enzyme are associated with neuromuscular dysfunction. (See, e.g., Corydon, M. J. et al. (1997) Mamm Genome 8(12):922-926.)
The discovery of a new human short-chain dehydrogenase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of immune disorders and cancer.
The invention features a substantially purified polypeptide, human short-chain dehydrogenase (HCSD), comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
Additionally, the invention provides a composition comprising a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO: 1 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing an immune disorder, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising substantially purified polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.